PROJECT SUMMARY In the nervous system, G protein-coupled receptors (GPCRs) sense neuromodulators to initiate intracellular signaling cascades that control a plethora of brain functions. Of particular importance are the opioid receptors which contribute to pain sensation, reward processing and mood regulation. Consistent with these roles, opioid receptors serve as major drug targets for a variety of disorders. Agonists of the mu-opioid receptor (MOR) are common analgesics and also have potential as antidepressants. However, a challenge with opioid- based treatment is the propensity for addiction, tolerance and dangerous side effects. Unfortunately, limitations in the precision of pharmacological approaches have hampered our ability to dissect the molecular, cellular and circuit level mechanisms of MOR-mediated disease treatment and develop improved therapeutic strategies. We previously established methodologies for optical control of GPCRs using photopharmacology, which we will adapt for the MOR in the R61 phase. In aim 1, we will develop a range of new photoswitchable compounds for the MOR using a combination of structure-based prediction, chemical synthesis and functional analysis. In aim 2, we will adapt these compounds to make photoswitchable orthogonal remotely-tethered ligands (PORTLs) which covalently attach to target receptors with a labeling tag, such as SNAP, CLIP or Halo. To enable targeting of native receptors, we will further extend our system to develop nanobody-photoswitch conjugates (NPCs) which bind to native receptors and deliver a PORTL for reversible optical control. NPCs can either be genetically- encoded to permit cell type-targeting or can be purified in vitro and directly applied to the sample in a gene-free approach. Together, this will provide a new, widely-applicable toolset for MORs while also developing an engineering framework for extension of this approach across different receptor types. In the R33 phase, we will harness our toolset in vivo to probe the basis of MOR-mediated antidepressant effects in mice (aim 3). We hypothesize that activation of MORs localized on interneurons of the medial prefrontal cortex (mPFC) induce disinhibition that leads to normalization of dysfunctional prefrontal circuits. PORTLs and NPCs will enable targeting to the cell types and brain regions of interest with sufficient spatiotemporal precision to probe the relationship between receptor activation and behavioral modulation. We will use behavioral assays of relevance to depression, as well as measures of reward and dependence with the expectation that targeted photo-activation can produce antidepressant effects with minimal side effects. To gain further mechanistic insight, we will perform slice electrophysiology and in vivo 2-photon calcium imaging to measure the effects of MOR activation on mPFC activity. This work will validate our toolset in vivo and provide a key step toward understanding the mechanism of MOR modulation for depression treatment.